Method for producing yeast expressed hpv types 6 and 16 capsid proteins

ABSTRACT

Mosaic VLPs of viral capsid proteins from different virus types are described, as are methods of making the same. Specifically, a diploid yeast strain that coexpresses the L1 and L2 capsid proteins of both HPV-6 and HPV-16 as mosaic VLPs is described. The mosaic VLPs induced the production of conformational antibodies against both L1 proteins upon administration to mice.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of Ser. No. 12/316,310, filedDec. 11, 2008, which is a Continuation of Ser. No. 11/404,063, filedApr. 13, 2006, now U.S. Pat. No. 7,494,787, which is a Divisional ofSer. No. 09/762,762, filed Apr. 9, 2001, now U.S. Pat. No. 7,112,330,which is a 371 filing of PCT/US99/18016, filed Aug. 13, 1999 whichclaims the benefit of Provisional Ser. No. 60/096,625, filed Aug. 14,1998, from which applications priority is claimed pursuant to 35 U.S.C.§§119/120, and which applications are incorporated herein by referencein their entireties.

FIELD OF THE INVENTION

The present invention is related to the production of mosaic virus-likeparticles comprising capsid proteins of human papilloma virus (HPV)types 6 and 16 capable of inducing immune response against both HPVtypes.

BACKGROUND OF THE INVENTION

A promising strategy to induce an immune response capable ofneutralizing papillomavirus (PV) infections is the use of virus capsidproteins as antigens. In the case of genital human papillomaviruses(HPVs), this approach was hampered by the lack of any in vivo or invitro source of sufficient amounts of native virus. In order to overcomethis problem, heterologous expression systems have been extensively usedto obtain large quantities of capsid proteins and to allow the analysisof their structural and immunological properties. Expression of themajor capsid protein late 1 (L1) from different PV types usingprokaryotic (25), baculovirus (21, 23, 37, 41, 42, 46), yeast (14, 18,19, 20, 29) and mammalian expression systems (15, 16, 51), demonstratedthat this protein can self-assemble into virus-like particles (VLPs).Coexpression of the minor capsid protein late 2 (L2) is not strictlynecessary to obtain VLPs, although its presence increases the efficiencyof particle formation (15, 22, 51) and induces anti-L2 neutralizingantibodies (32). The L1 and L2 VLPs appear similar to native virions byelectron microscopy (EM). The use of different animal models has shownthat VLPs can be very efficient at inducing a protective immuneresponse.

VLPs meet many of the criteria which make them ideal surrogates ofnative virions. They resemble infectious particles by ultrastructuralanalysis (16), elicit virus neutralizing antibodies and bind to theputative receptor on the surface of mammalian cells (28, 31, 33, 44,47). Most notably, the results obtained with animal models demonstratedthat prophylactic immunization with VLPs can be very effective in vivo.Cottontail rabbits, calves and dogs immunized with L1 VLPs wereprotected from subsequent challenge with the homologous PV (20, 23, 41)and passive transfer of immune sera conferred protection to naiveanimals (20, 41), indicating that an antibody-mediated response plays amajor role in preventing virus infection.

Studies with infectious HPV virions, as well as VLPs of different HPVtypes, strongly suggested, however, that the immune response ispredominantly type-specific. Further, the efficacy of VLP-based anti-HPVvaccine candidates cannot be evaluated in animals since these virusesexhibit a high degree of species specificity. Antibody-mediated virusneutralization has been therefore studied using either in vitro assays(35, 40) or xenograft systems which allow propagation of infectiousvirus of specific HPV types (1, 2, 5, 6, 24). The primary conclusionwhich could be drawn from these experiments was that immunization withHPV VLPs evokes a neutralizing immune response which is predominantlytype-specific (6, 7, 34, 35, 36, 48).

Cross-neutralization has been reported between HPV-6 and HPV-11 (92%amino acid sequence identity) (8) and between HPV-16 and HPV-33 (80%amino acid sequence identity) (48). This may indicate the existence ofsome correlation between protein sequences and structural similaritiesthat could possibly be relevant for the mechanism of capsid assembly. Onthe basis of these considerations, however, the concept that HPV-6 andHPV-16 L1 proteins may coassemble is not obvious, since the two virusesbelong to phylogenetically more distant groups (3, 45) and exhibit alower (67%) L1 amino acid sequence identity.

Further, while envelope proteins of viruses belonging to very differentfamilies can be incorporated into the same envelope (50), nucleocapsidprotein mixing appears to be much more restricted. Mixed core particlesbetween Moloney murine leukaemia virus (MuLV) and human immunodeficiencyvirus (HIV) have been obtained but only when artificial chimeric Gagprecursors, containing both HIV and MuLV determinants are coexpressedwith wild-type MuLV Gag proteins (10). By using a yeast two-hybridsystem based on GAL4-Gag fusion protein expression plasmids, Franke etal. were able to show that the ability of two heterologous Gag proteinsto multimerize was correlated with the genetic relatedness between them(13).

Mixed capsid formation between wild-type Gag proteins has not beenreported so far. In the case of the hepadnavirus core (C) protein, Changet al. (4) have shown that an epitope-tagged truncated hepatitis B virus(HBV) C polypeptide could coassemble in Xenopus oocytes with woodchuckhepatitis virus (WHV) and ground squirrel hepatitis virus (GSHV) Cproteins but not with that of duck hepatitis B virus (DHBV). This resultwas not unexpected since the two core protein sequences have divergedsignificantly and do not show immunological cross-reactivity. Whencoassembly of C polypeptides of HBV, WHV and GSHV occurred, formation ofmixed capsids resulted from the aggregation of different species ofhomodimers (4).

Several reports have discussed the importance of disulfide bonds for theintegrity of native bovine papillomavirus type 1 (BPV-1) virions (26)and VLP structures (25, 38, 39). Li et al. (26) have also shown that thecysteine 424 mutant (C424) of HPV-11 L1 in the carboxy-terminal domainthat has been identified as critical for capsid formation (25), is stillable to form capsomeres but not VLPs, indicating that this residue maybe involved in interpentamer bonding. The essential role of disulfidebonds has been confirmed by a single point mutation of either C176 orC427 in HPV-33 L1 (C428 in HPV-18 L1), which converts all VLP trimersinto monomers, allowing capsomere formation but not VLP assembly (39).

It has been recently proved that; by using an in vitro infection systemand a sensitive reverse transcriptase PCR-based assay (RT-PCR), antiserato HPV-6 VLPs are not able to neutralize authentic HPV-16 virions (48).Since cysteine residues corresponding to those described as involved indisulfide bonding above are conserved in the HPV-6 and HPV-16 L1proteins, we hypothesized that mosaic VLPs could either result fromintra-capsomeric or inter-capsomeric association of the two proteinsand/or from interaction between type-specific subsets of capsomeres.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a method for producingmosaic virus like particles comprising the capsid proteins from at leasttwo types of viruses, preferably animal, more preferably HPV. In apreferred aspect, the capsid proteins are from HPV types 6 and 16. In afurther preferred aspect, the capsid proteins are L1 and L2 from HPVtypes 6 and 16.

In a further aspect, the present invention relates to vectors and hostsfor expressing the capsid proteins of at least two types of viruses,preferably animal, more preferably HPV. In a preferred aspect, thecapsid proteins are from HPV types 6 and 16. In a further preferredaspect, the capsid proteins are L1 and L2 from HPV types 6 and 16. In afurther preferred aspect, the present invention relates to a diploidyeast strain that coexpresses the L1 and L2 capsid proteins of HPV-6 andHPV-16 as mosaic VLPs.

In another aspect, the present invention relates to a method forinducing an immune response against more than one type of virus usingmosaic VLPs comprising capsid proteins from each virus type. In apreferred aspect, the mosaic VLPs comprise capsid proteins from animalviruses, more preferably HPV, most preferably HPV types 6 and 16. In afather preferred aspect, the mosaic VLPs comprise the L1 and L2 capsidproteins from HPV types 6 and 16.

In still another aspect, the present invention relates to an immunogenicvirus like particle comprising capsid proteins from different types ofviruses, preferably animal, more preferably HPV, most preferably HPVtypes 6 and 16. In a further preferred aspect, the mosaic VLPs comprisethe L1 and L2 capsid proteins from HPV types 6 and 16.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the construction of the pBS-6L1 plasmid.

FIG. 2 depicts the recombinant PCR performed in constructing the pBS-6L1plasmid.

FIG. 3 depicts a Western blot analysis of cell extracts from yeaststrains expressing HPV-6 and HPV-16 capsid proteins. Equivalent amountsof total cell extracts from the parental JSC310 strain (lanes 1) anddifferent recombinant strains (lanes 2 and 3) were separated by 10%SDS-PAGE, electrotransferred to nitrocellulose filters and incubatedwith the H6.C6 (a) or the H16.H5 (c) type-specific anti-L1 Mabs, andwith HPV-6L2 (b) or HPV-16L2 (d) antisera. Lanes 2a and 2c: JSC310-6L1epi; lanes 3a and 3c: JSC310-16L1 epi; lanes 2b and 2d: JSC310-6L2epi;lanes 3b and 3d; JSC310-16L2epi. Molecular mass standards (in kDa) areindicated. This multipanel figure and those which follow have beenassembled by using Photoshop 4.0 and FreeHand 7.0 programs forMacintosh.

FIG. 4 is a schematic representation of the yeast integrative plasmidsYIpAde (a) and YIpLys-L2 (b) vectors. The continuous lines represent pUCvector sequences. The empty box in (a) represents the adenine 2 genesequence. The black boxes in (b) represent lysine 2 gene fragments, thegrey box represents the L2 gene, the empty boxes represent the ADH2/GAPhybrid promoter and the MFα gene transcriptional termination sequence.The arrow in the L2 box indicates the 5′-3′ orientation of the codingsequence. Relevant restriction sites are indicated.

FIG. 5 depicts a Western blot analysis of cellular extracts fromrecombinant haploid and diploid yeast strains. Total cell extracts wereseparated by 10% SDS-PAGE, electrotransferred to nitrocellulose filtersand incubated with anti-HPV-6 L1 (a) and anti-HPV-16 L1 (c) Mabs andwith HPV-6 L2 (b) and HPV-16 L2 (d) antisera. Lanes 1: AB110-6L1/16L2;lanes 2: JSC310-16L1/6L2; lanes 3: AB/JS-4L; lanes 4: JSC310-6L2epi;lanes 5: JSC310-16L2epi. Arrows in (b) and (d) indicate the bandscorresponding to the L2 proteins. Molecular mass standards (in kDa) areindicated.

FIG. 6 depicts an analysis of fractions from CsCl gradient sedimentationof AB/JS-4L cell extract. (A) Aliquots from fractions 1 to 9 wereblotted onto nitrocellulose filters using either (a and c) denaturingand reducing (D) or (b and d) nondenaturing and nonreducing (N)conditions. The filters were incubated with the type-specific anti-L1H6.C6 (a) and H16.H5 (c) Mabs, and with the conformationally dependenttype-specific anti-L1 H6.B10.5 (b) and H16.V5 (d) Mabs. As a control,the anti-HPV-6 and HPV-16 L1 conformational Mabs were incubated withCsCl purified VLPs (e) blotted under either denaturing or nondenaturingconditions. The arrows in A indicate fraction no. 5. (B) Aliquots offraction no. 5 were subjected to SDS-PAGE, electroblotted onnitrocellulose filters and incubated either with HPV-6 L2 (lane 3a) orHPV-16 L2 (lane 3b) antiserum. As a control, total cell extracts fromthe JSC310-6L2epi (lanes 1) and JSC310-16L2epi (lanes 2) strains wereused. Molecular mass standards (in kDa) are indicated. Arrows indicatebands corresponding to the L2 proteins.

FIG. 7 depicts an electron microscope (EM) analysis of CsCl purifiedVLPs. HPV-6 (a), HPV-16 (b) and HPV-6/16 VLPs were adsorbed ontoFormvar-carbon coated grids, stained with 4% uranyl acetate and examinedunder a Zeiss EM10C microscope at a magnification of ×100,000 (Bar=100nm).

FIG. 8 depicts a Western blot analysis of immunoprecipitated VLPs. CsClbanded VLPs from the AB/JS-4L diploid strain were immunoprecipitatedwith the anti-HPV-6 L1 conformationally dependent H6.B10.5 Mab. Theimmunoprecipitated proteins were separated using a 15 centimeter (cm)long 10% polyacrylamide SDS-gel, electroblotted on nitrocellulosemembrane and incubated either with the anti-HPV-6 L1 specific H6.C6 Mab(a) or with the anti-HPV-16 L1 specific H16.H5 Mab (b). Controlreactions, including either VLPs or the conformational Mab only, wereset up and processed under identical experimental conditions. Lane 1:VLPs incubated overnight without the Mab; lane 2: Mab incubatedovernight; lane 3: VLPs incubated overnight with the H6.B10.5conformational Mab; lane 4: total cell extract from the JSC310-6L1epistrain; lane 5: total cell extract from the JSC310-16L1 epi strain.Arrows indicate a conformational Mab-derived band (A), the L1 bands (B)and a protein A Sepharose-derived band (C).

FIG. 9 depicts a characterization of sera derived from mice immunizedwith HPV-6, HPV-16 and mosaic VLPs. (A) Comparable amounts of HPV-6(lanes 1), HPV-16 (lanes 2) and mosaic VLPs (lanes 3) were separated onSDS-PAGE and immunoblotted with antisera from mice immunized with HPV-6VLPs (a) HPV-16 VLPs (b) and mosaic VLPs (c). (B) Comparable amounts ofHPV-6 and HPV-16 VLPs were dot-blotted under denaturing and reducing (D)and nondenaturing and nonreducing (N) conditions and incubated with theS16 antiserum of a mice immunized with mosaic VLPs.

DETAILED DESCRIPTION OF THE INVENTION

To test the possibility of inducing antibodies against multiple HPVtypes, we have generated a recombinant yeast diploid strain thatcoexpresses the HPV-6 and HPV-16 L1 and L2 genes. HPV-6/16 mosaic VLPswere purified from the cell lysate and used as antigens to immunizemice. The data presented below supports the formation of mosaic VLPscomprising all four proteins. The immunoprecipitation experimentstrongly suggests that the CsCl purified VLPs represent the result of areciprocal interaction of the two L1 proteins, rather than the simplecoexistence of different VLP types. The fact that the L2 proteins arepresent in the same CsCl fractions favors the hypothesis that they areincorporated into the VLPs as well, since the L2 protein alone does notband in a CsCl gradient at the same density as L1 VLPs (22). Further,antisera able to recognize conformational epitopes of both L1 proteinswere obtained. Although it remains to be confirmed that the immuneresponse elicited by HPV-6/16 VLPs can neutralize the two viruses, thedata herein supports using mosaic VLPs to immunize against a broaderspectrum of virus types.

A yeast expression system as herein disclosed is preferred. Differentlaboratories have observed that a Saccharomyces cerevisiae expressionsystem can be successfully used to easily purify PV VLPs (14, 18) whichare highly efficient at inducing a protective immune response in animalmodels (20). Yeast-expressed VLPs are able to elicit a specific immuneresponse not only at systemic but also at mucosal level. Lowe et al.have reported the generation of IgG neutralizing antibodies in the seraand genital secretions of African green monkeys immunizedintramuscularly with HPV-11 VLPs, adsorbed to aluminum adjuvant (27).Greer et al. have observed the induction of anti-L1 specific IgG and IgAantibodies in the sera and genital secretions of mice immunizedintranasally with HPV-6 VLPs, adjuvanted either with E. coli heat-labileenterotoxin (LT) or with a LT-derived non toxic mutant (14). Further,yeast expression affords the potential to scale-up to thousands ofliters at relatively low cost and many yeast-derived products for humanuse are already market approved due to their safety.

To express the HPV-6 and HPV-16 L1 and L2 genes in the same yeast cell,we generated a S. cerevisiae diploid strain by mating two haploidstrains, each expressing two of the four capsid proteins. In order toobtain expression of the heterologous genes under identical cultureconditions, each of them was cloned into the same expression cassettebased on the ADH2/GAP glucose-repressible hybrid promoter and theT_(MFα) transcriptional termination sequence. The HPV-6 and HPV-16 L1proteins were expressed by means of the episomal expression vectorpBS24.1. Expression of the HPV-6 and HPV-16 L2 proteins was insteadobtained by cloning the expression cassette into an integrative plasmidsuitable for insertion into the lys2 locus of the haploid strain genome(FIG. 4 b). As a consequence of this cloning strategy, the L1 and L2gene copy numbers in the haploid strains were different and thisresulted in higher expression levels of the L1 proteins. This shouldresemble the ratio of L1 to L2 observed in native HPV virions, which hasbeen estimated over a range from 5:1 to 30:1 (25). Table 1 lists theparental yeast strains used, the two recombinant haploid strainsobtained and the diploid strain resulting from the mating.

TABLE 1 List of parental and recombinant yeast strains with genotypesand HPV expressed genes Episomal Integrated Yeast strain Genotype HPVgene HPVgene JSC310 MATa leu2-3 ura3-52 prb1-1122 pep4-3 prc1-407adr1::DM15 cir ° AB110 MATα leu2-3-112 ura3-52 pep4-3 his4-580 cir °JSC310-6L1epi MATa prb1-1122 pep4-3 prc1-407 adr1::DM15 cir ° 6L1JSC310-16L1epi MATa prb1-1122 pep4-3 prc1-407 adr1::DM15 cir ° 16L1JSC310-6L2epi MATa prb1-1122 pep4-3 prc1-407 adr1::DM15 cir ° 6L2JSC310-16L2epi MATa prb1-1122 pep4-3 prc1-407 adr1::DM15 cir ° 16L2JSC310-6L2int MATa leu2-3 ura3-52 prb1-1122 lys2 pep4-3 prc1-407adr1::DM15 cir ° 6L2 AB110-16L2int MATα leu2-3-112 ura3-52 pep4-3 lys2his4-580 cir ° 16L2 JSC310-16L1/6L2 MATa prb1-1122 lys2 prc1-407 pep4-3ade2 adr1::DM15 cir ° 16L1 6L2 AB110-6L1/16L2 MATα pep4-3 lys2 his4-580cir ° 6L1 16L2 AB/JSC-4L MATa/MATα PRB1/prb1-1122 lys2/lys2PRC1/prc1-407 pep4-3/pep4-3 6L1-16L1 6L2- 16L2 HIS4/his4-580ADR1/adr1::DM15 cir °

As used herein, the term “mosaic VLP” refers to a VLP comprising capsidproteins from more than one type of virus. VLPs which result from intra-and/or inter-capsomeric association of the proteins are included.

As used herein, the term “type” in reference to viruses includes viruses(animal and plant) within the same family, group, or genus as well asviruses in different families, groups, or genuses.

As used herein, the term “non-integrative” in reference to a vectorindicates that the vector does not integrate into the host DNA.

Yeast strains. The Saccharomyces cerevisiae haploid strains used wereJSC310 (MATa, leu2-3, ura3-52, prb1-1122, pep4-3, prc1-407, adr1::DM15,cir °) (17) and AB110 (MATα, leu2-3-112, ura3-52, pep4-3, his4-580, cir°) (43), provided by Vicky Hines (Chiron Corporation, Emeryville,Calif., USA).

Monoclonal and polyclonal antibodies. The H6.C6 and H16.H5 monoclonalantibodies (Mabs), which bind to denatured HPV-6 and HPV-16 L1 proteins,respectively, in addition to the H6.B 10.5 and H16.V5 Mabs, specific forHPV-6 and HPV-16 intact VLPs, have been reported by Christensen et al.(8, 9). For Western blot analysis, these Mabs were used at 1:3000dilution with a 4° C. overnight incubation. HPV-16 L2 rabbit antiserumwas a gift of Lutz Gissmann (DKFZ, Heidelberg, Germany), while HPV-6 L2rabbit antisera were kindly provided by Denise Galloway (Fred HutchinsonCancer Research Center Seattle, Wash.) and Robert C. Rose (University ofRochester, N.Y.). All the antisera were used at 1:3000-5000 dilutionwith a 4° C. overnight incubation. Anti-rabbit and anti-mouseperoxidase-conjugated antibodies were from Biosource International(Camarillo, Calif.) and were used at 1:5000 dilution at room temperaturefor 1.5 hours.

Example 1 HPV Type-Specific Detection of Capsid Proteins Expressed inYeast

A single yeast strain which could express the four HPV-6 and HPV-16 L1and L2 capsid proteins was prepared. A necessary tool in achieving thiswas the availability of antibodies which reacted specifically orpreferentially with the L1 or the L2 protein of only one HPV type. TheHPV-6 and HPV-16 L1 and L2 genes were cloned in the episomal vectorpBS24.1 (see Example 2 below) and expressed in the S. cerevisiae strainJSC310 to test the type specificity of the available antibodies. FIG. 3shows the results of a Western blot analysis of total cell extractsprepared from the recombinant strains incubated with specific anti-HPV-6(a) or HPV-16 (c) L1 Mabs and with HPV-6 (b) or HPV-16 (d) L2 antisera.In all cases HPV type-specific bands were detected, although a weakcross-reactivity could be seen for both the L2 antisera. While the HPV-6and HPV-16 L1 Mabs identified proteins with the expected molecularweight of about 55 kilodalton (kDa), the L2 proteins, as previouslyreported (11, 12), showed an electrophoretic mobility corresponding toapproximately 72-75 kDa, instead of the 55 kDa predicted on the basis oftheir amino acid sequences.

Example 2 Construction of Recombinant Plasmids

DNA fragments encoding the HPV proteins were obtained from availablerecombinant plasmids, either by restriction enzyme digestion or by PCRamplification (Expand High Fidelity PCR System, Boehringer Mannheim),and they were completely sequenced using an Applied Biosystem (Norvalk,CELLTECH, USA) model 373 DNA sequencer.

The episomal yeast expression vector pBS24.1, a yeast “shuttle” vector(17 and Philip J. Barr, Chiron Corporation, Emeryville, Calif., USA),containing the leucine 2 (Leu2) and uracil 3 (Ura3) selectable genes wasused. In this instance, it was obtained by digesting an availablepBS24.1αt6E7 plasmid with Bam HI and Sal I. The pBS24.1αt6E7 plasmid wasprepared for the yeast expression of the HPV-6E7 antigen in a secretedform.

The pBS-6L1 plasmid, expressing the HPV-6 L1 protein under the controlof the alcohol-dehydrogenase-2-glyceraldehyde-3-phosphate-dehydrogenase(ADH2/GAP) glucose repressible promoter (J. Shuster, Chiron Corporation,Emeryville, Calif., USA) and the mating type alpha factor genetranscriptional termination sequence (T_(MFα)) was derived from thepBS24.1 plasmid as follows.

The plasmid pBS-6L1 is a yeast expression vector which contains theHPV-6L1 under the control of the ADH2\GAP promoter cloned into BAM HIand Sal I sites of the vector pBS24.1. The vector pBS24.1 contains theα-factor terminator, therefore an “expression cassette” for HPV-6 L1 isobtained. The “expression cassette” for HPV-6L1 consists of thefollowing sequences fused together (from 5′ to 3′): ADH2\GAP hybridpromoter, HPV-6L1 gene, and α-factor terminator. At the end of thecloning procedures the above “expression cassette” was obtained into thepBS24.1 (17). The vector pBS24.1 may be replicated both in Escherichiacoli and in Saccharomyces cerevisiae since it contains PBR322 sequences(including the origin of replication and the ampicillin resistance gene)and the complete 2μ sequences (including the origin of replication). Italso contains the yeast URA3 gene and the yeast LEU2 gene.

A summary of the construction of plasmid pBS24.1-A/G-6L1 is presentedschematically in FIG. 1. Due to the lack of suitable restriction sites,the fusion between the glucose repressible ADH2\GAP promoter and the L1ORF has been obtained by means of recombinant PCR. The 1-563 bp segmentof the hybrid promoter (1113 bp long) is derived from GAGαt6E7 plasmidwhilst the 564-1113 bp are derived from PCR amplification of Gga plasmid(see below). The 1-115 bp segment of L1 sequence (1503 bp long) isderived from PCR amplification of the pAcC13-6L1 plasmid (Greer et al.,J. Clin. Microbiology, 2058-2063, 1995 and Munemitsu et al., Mol. CellBiol., 10:5977-5982, 1990), whilst the 116-1503 bp segment is derivedfrom pAcC13-6L1 plasmid directly. The DNA sequence of HPV 6 is reportedin Schwarz et al., EMBO J., 2:2341-2348, 1983.

The GAGαt6E7 plasmid is a derivative of pGEM-3z (Promega) vector inwhich the following sequence was constructed (from 5′ to 3′): ADH2\GAPpromoter, an α-factor derived leader sequence, and the HPV-6E7 codingsequence. The GAGαt6E7 plasmid was digested with Bcl I and Xba I. TheDH5α derived plasmid DNA could not be cut with Bcl I because the DH5αcells are dam+, but the Bcl I enzyme is inhibited by overlapping dammethylation; in order to obtain a Bcl I digestible DNA the plasmid wastransformed in the dam-JM110 E. coli cells (Stratagene). The JM110derived plasmid was digested with Bcl I and Xba I, the fragmentcontaining the vector and the 5′ half of the ADH2\GAP promoter was gelpurified and set aside for further ligation.

The pAcC13-6L1 plasmid was digested with Xba I, the insert was gelpurified and set aside for ligation. The Xba I insert consisted in theL1 sequence from bp 115 to the end of the sequence, including the stopcodon.

The recombinant PCR is schematically represented in FIG. 2. Thesequences of the primers are listed below.

SEQ ID NO: 1 RP5 5′-ACTGATAGTTTGATCAAAGGGGCAAAACGTAGGGGC-3′ SEQ ID NO: 2RP6 5′-GTCGCTAGGCCGCCACATGGTGTTTGTTTATGTGTG-3′ SEQ ID NO: 3RP7 5′-AAACACACATAAACAAACACCATGTGGCGGCCTAGC-3′ SEQ ID NO: 4RP8 5′-GCAGTCACCACCCTGTACAGGTGTATTAGTACACTG-3′

A first PCR was performed using the RP5 and RP6 primers and the Ggaplasmid DNA as template. The Gga plasmid is a pGEM-3z plasmid derivativeobtained in the context of the previous procedures for the HPV-6E7cloning in yeast and contains the ADH2 \GAP promoter. The goal of thisfirst PCR was to obtain the 563-1113 bp portion (3′ half) of theADH2\GAP promoter. The RP5 primer overlapped a Bcl I site. A second PCRwas performed using the RP7 and RP8 primers and the pAcC6L1 (Greer etal., 1995) plasmid as template. The goal of this second PCR was toamplify the 5′ end of the L1 sequence from the initiation codon to theby 543. The amplified fragment would contain an Xba I site at position115. The RP6 and RP7 primers were designed in such a way that the 3′ endof the first PCR product would anneal to the 5′ end of the second PCRproduct. A third PCR was performed by mixing the first and secondamplimers and the external primers RP5 and RP8. During this PCR ajoining between first and second amplimers would happen and also anamplification of the joined product.

The expected 1126 bp product of the third PCR was predicted to consistin the 563-1113 (3′ half) sequence of the ADH2 \GAP promoter joined tothe 1-530 (5′ end) sequence of the HPV-6L1 ORF. The final PCR productwould have a Bcl I site at the 5′ end and an Xba I site in the L1portion of the sequence at position 115. The third PCR product wasdigested with Bcl I and Xba I and gel purified. The fragment containingthe pGEM-3z vector and the 5′ half of the promoter coming from the BclI-Xba I digestion of the GAGαt6E7 plasmid was ligated with the Bcl I-XbaI digested recombinant PCR product and to the L1 insert coming from theXba I digestion of pAcC13-6L1 plasmid.

After transformation into DH5α cells, several transformants wereobtained. The miniprep DNAs from 14 transformants were digested usingEco RI. The Eco RI enzyme was chosen because by using this enzyme it hasbeen possible to verify both the expected molecular sizes and thecorrect orientation of the 6L1 fragment. The 6L1 fragment had identicalextremities (such as Xba I), therefore the probability for the fragmentto assume an opposite orientation was 50%. By using Eco RI the plasmidDNA of the right clones should give two fragments, 2600 and 2700 bplong. The miniprep DNA of the n° 8 clone gave a single band on a firstgel but by running the gel much more was possible to resolve the 2600and 2700 bp fragments. Also using Sph I it was possible to have afurther indication that the clone n° 8 was good. It was, thus, assumedthat the clone n° 8 contained the correct pGAG-6L1 plasmid consisting inthe pGEM-3z vector containing the HPV-6 L1 sequence under the control ofthe ADH2\GAP promoter.

The ADH2\GAP-HPV-6L1 insert was excised from pGAG-6L1 plasmid bydigesting with Bam HI and Sal I, the insert was gel purified and setaside for further ligation. The promoter-L1 fragment and the pBS24.1vector were ligated and the product of the reaction was transformed intoDH5α cells. The miniprep DNAs from 5 transformants were analyzed bydigesting the Bam HI and Sal I and the clones A, B, C, and E wereselected as good clones exhibiting the right molecular weight pattern.

A clone was transformed in JSC310 strain of Saccharomyces cerevisiae bymeans of electroporation and the cells were plated on URA-plates.Selected transformants were picked from URA-plates and streaked onLEU-plates. Single colonies from LEU-plates were inoculated inLEU-medium. Four clones grown in LEU-medium were reinoculated in YEPDmedium. Cell pellets from the four JSC310-6L1 clones, A, B, C and D werefrozen at −20° C. after 24 and 48 hours of growth in YEPD medium onpurpose to check L1 protein expression. Glycerol batches of the fourclones were stored at −80° C.

The 6L1 yeast cell pellets were glass beads extracted, soluble andinsoluble extracts were separated by means of centrifugation andprepared for SDS-PAGE analysis. Extracts from a strain not containingthe pBS-6L1 plasmid (JSC310 cells transformed with pAB24 vector) werealso prepared as a negative control. In Coomassie strained gel and inwestern immunoblot an induced band exhibiting the expected molecularweight was visible. A comparison of the HPV-6L1 expressed in the yeastJSC310 strain and the same antigen expressed in insect cells showed thatthe two antigens have similar molecular weight.

The DNA portion of the L1 gene deriving from recombinant PCR (bp 1-115)has been sequenced using the following primer:

5′ TAGTTTTAAAACACCAA 3′. SEQ ID NO: 12The primer annealed at the 3′ end of the ADH2\GAP promoter, at position−37 from the L1 start codon. The pGAG-6L1 plasmid (pGEM-3z containingthe ADH2\GAP promoter fused to the L1 sequence) was used as template. Bysequencing it was established that no errors occurred during therecombinant PCR manipulations nor in the cloning steps.

To construct the YIpAde integrative plasmid, a 1,059 bp XbaI genomic DNAfragment of the S. cerevisiae adenine 2 gene (Ade2) was amplified byusing the PCR oligonucleotide primers 5′ AdeE(5′-GCGGCGAATTCTAGAACAGTTGGTATATTAG-3′ SEQ ID NO:5, inserting an EcoRIsite) and 3′ AdeP (5′GCGGCCTGCAGGGTCTAGACTCTTTTCCATATA-3′ SEQ ID NO:6,inserting a PstI site). The amplified DNA fragment was cloned intoplasmid pUC8 digested with EcoRI and PstI and the XbaI sites, includedin the amplified DNA fragment, were used to excise the insert for yeasttransformation. To obtain the integrative YIpLys-L2 expression plasmids,a 1,318 bp genomic DNA fragment of the S. cerevisiae lysine 2 (Lys2)gene was amplified by using the PCR oligonucleotide primers 5′LysE(5′-GCGGAATTCCACTAGTAATTACA-3′ SEQ ID NO:7, inserting an EcoRI site) and3′LysH (5′-GATGTAAGCTTCTACTAGTTGA-3′ SEQ ID NO:8, inserting a HindIIIsite). The amplified DNA fragment was then inserted into pUC8(derivatives readily available from commercial sources, e.g., Promega)digested with EcoRI and HindIII, generating a plasmid named YIpLys. ABamHI DNA fragment from pSI3 vector (Isabel Zaror, Chiron Corporation,Emeryville, Calif., USA, pBR322 backbone, ADH2/GAP promoter, SODprotein, and T_(MFα)), including the ADH2/GAP promoter, the humansuperoxide dismutase (SOD) gene and the T_(MFα) transcriptionaltermination sequence, was cloned into the single BglII restriction sitein the Lys2 gene sequence of YIpLys, obtaining a plasmid namedYIpLys-SOD. The YIpLys-6L2 plasmid was derived from YIpLys-SOD replacingthe NcoI-SalI DNA fragment encoding the SOD gene with the NcoI-SalI DNAfragment from pGEM3z-6L2 (Kent Thudium, Chiron Corporation, Emeryville,Calif., USA) encoding the HPV-6b L2 open reading frame (ORF). Toconstruct the YIpLys-16L2 plasmid, the L2 gene was amplified from thecloned HPV-16 genomic DNA (kindly provided in this instance by Dennis J.McCance, University of Rochester, N.Y.) by using the PCR oligonucleotideprimers DT-5′L2 (5′-CGACACAAACGTTCTGCAA-3′ SEQ ID NO:9) and DT-3′L2(5′-ATTAGTCGACCTAGGCAGCCAAGAGACATC-3′ SEQ ID NO:10), including thetranslation termination codon and a SalI site. The DNA fragment obtainedwas digested with SalI and cloned into YIpLys-SOD from which the SODcoding sequence had been removed by digestion with NcoI, filling-in withKlenow enzyme and digestion with SalI.

The pBS-6L2 and pBS-16L2 episomal expression plasmids were obtained byreplacing a SacI-SalI DNA fragment from pBS-6L1, including part of theADH2/GAP promoter and the entire HPV-6b L1 ORF, with SacI-SalI DNAfragments, derived from either YIpLys-6L2 or YIpLys-16L2, including thecorresponding promoter region and the L2 ORF.

To construct the pBS-16L1 episomal expression plasmid, the L1 gene wasamplified from cloned HPV-16 genomic DNA by using the PCRoligonucleotide primers DT-5′L1 (5′-TCTCTTGGCTGCCTAGTGAGGCCA-3′ SEQ IDNO:11) and DT-3′L1 (5′-CTAGTAATGTCGACTTACAGCTTACGTTTTTTGCG-3′ SEQ IDNO:12), comprising the translational termination codon and a SalI site.The amplified DNA fragment was purified from agarose gel and cloned intoblunt-ended pSI3 vector from which the SOD gene had been previouslyremoved by digestion with NcoI and SalI restriction enzymes andfilling-in with Klenow enzyme. From this intermediate construct, aSacI-SalI DNA fragment, including part of the ADH2/GAP promoter and theHPV-16L1 ORF, was purified and used to replace the correspondingSacI-SalI DNA fragment in pBS-6L1.

Example 3 Generation of Recombinant Yeast Strains

The strains JSC310-6L1 epi (14), JSC310-16L1epi, JSC310-6L2epi andJSC310-16L2epi, expressing the four capsid proteins by means of episomalvectors, were obtained by transformation of the parental JSC310 strainwith the expression plasmids pBS-6L1 (14), pBS-16L1, pBS-6L2 andpBS-16L2.

The JSC310-6L2int and the AB110-16L2int strains were obtained using thefollowing experimental approach. Competent yeast cells werecotransformed with 5 μg of EcoRI-HindIII digested YIpLys-6L2 orYIpLys-16L2 integrative plasmid and 1 μg of pBS24.1 episomal vector toallow the selection of transformants. Different clones were tested forgrowth onto plates of minimal medium (MM) supplemented with α-adipate toselect mutants with an inactivated Lys2 gene (49). Correct integrationinto the lys2 locus was verified by PCR analysis by using pairs ofoligonucleotide primers complementary to sequences within the expressioncassette and the genomic portion of the Lys2 gene. Among the coloniesexpressing the L2 protein, one was chosen, cured of the pBS24.1 plasmidand tested for the inability to grow in the absence of uracil andleucine. Introduction of the episomal L1 expressing vectors into thesestrains was carried out following two different strategies.AB110-16L2int was transformed with the pBS-6L1 expression plasmid andselection of transformants on MM plates without leucine and uracilallowed the isolation of the haploid strain AB110-6L1/16L2. TheJSC310-6L2int strain was instead cotransformed with the pBS-16L1expression vector and with the XbaI digested YIpAde integrative plasmid.Transformants grown on selective plates were plated on complete yeastextract-peptone medium (YEP) and allowed to grow at 30° C. for 3-4 daysuntil colonies (1-2%) developed a red color due to disruption of theade2 locus (52). One of the clones, which showed correct integrationinto the ade2 locus by PCR and L1 and L2 expression by Western blotanalysis, was designated JSC310-16L1/6L2.

Generation of the AB/JSC-4L diploid strain was obtained by mixingcultures, in YEP medium containing 5% glucose, of the two haploidstrains, AB110-6L1/16L2 and JSC310-16L1/6L2. Selection of the AB/JSC-4Ldiploid strain required an additional genetic marker in the haploidJSC310-6L2int strain. This was obtained inactivating the endogenous Ade2gene by means of the integration plasmid represented in FIG. 4 a.Diploid cells were selected onto MM plates lacking histidine andadenine.

Expression of the four proteins in the haploid strains and in the strainresulting from their mating was evaluated by Western blot analysis. FIG.5 shows the results of such experiments demonstrating that both thehaploid strains AB110-6L1/16L2 (a and d, lanes 1) and JSC310-16L1/6L2 (band c, lanes 2) expressed the heterologous genes and that the expressionof all four proteins was stably maintained in the resulting AB/JS-4Ldiploid strain (a, b, c and d, lanes 3).

Example 4 Preparation of VLPs

Parental east strains were grown in complete YEP medium. Strainstransformed with episomal vectors were first cultured inleucine-deficient MM medium with 4% glucose until they reached midlogphase. Expression of the genes under the control of the ADH2/GAPglucose-repressible promoter was induced by diluting these cultures 1:50into YEP complete medium and culturing the cells at 30° C. for 2-3 days.Total cell extracts were prepared from 3.5 optical densities (OD) ofyeast cell cultures grown to approximately OD₆₀₀=20. Cells were lysedwith a 10 minute incubation on ice in 0.24 N NaOH and 0.96%β-mercaptoethanol, followed by trichloroacetic acid (TCA) precipitation,ice cold acetone washing and final suspension of the protein pellet in100 μl of protein loading buffer. To carry out dot-blot experimentswhere preservation of L1 conformation was necessary, yeast cells werecollected, washed, suspended in phosphate-buffered saline (PBS, pH 7.5)and disrupted by vortexing five times for 1 minute in the presence ofglass beads (425-600 μm, Sigma).

Frozen yeast cell pellets were thawed in buffer containing 0.1 MTris-HCl (pH 7.5), 0.15 M NaCl, 2 mM MgCl₂ and 1 mM EGTA (#E3889, SigmaChemical Co.) and Complete™Protease Inhibitors (#1-697-498, BoehringerMannheim). Cells were disrupted by vortexing twice for 10 minutes, witha 5 minute interval on ice, in the presence of glass beads (0.5 ml beadsper ml of cell suspension) using a VWRbrand Multi-tube vortexer (VWRScientific Product). Cellular debris was removed by a 20 minutecentrifugation at 2000×g. The supernatants were then centrifuged througha 40% (w/w) sucrose cushion (2 hour centrifugation at 100,000×g). Theresulting pellets were suspended in PBS, applied to a pre-formed CsClgradient (1.17-1.57 g/ml) and centrifuged for 24 hours at 285,000×g. Thegradients were fractionated and aliquots from each fraction weresubjected to Western blot analysis with type-specific anti-L1 andanti-L2 antibodies. Peak fractions were pooled and dialyzed against PBS.Total protein concentration was determined by BCA™ Protein Assay Reagent(#23225, Pierce Chemicals).

Example 5 Characterization of VLPs

Proteins were analyzed by denaturing sodium-dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE, 10% polyacrylamide) andWestern blotting onto nitrocellulose membrane (pore size 0.45 μm, MSI,Westborough, Mass. USA) according to standard protocols. Dot-blotanalysis of denatured and reduced VLPs was carried out boiling theprotein samples for 5 minutes in the presence of dithiothreitol (DTT)before applying them to nitrocellulose filters using a bio-dot apparatus(Biorad). When native VLP structure had to be maintained, VLPs in PBSwere applied to the membrane without boiling and in the absence of DTT.Reaction with HPV-specific antibodies was detected using the EnhancedChemiluminescence (ECL) Western blotting reagent (Amersham) andHyperfilm ECL (Amersham).

Specifically, the cell extract from the diploid strain was subjected toCsCl gradient sedimentation and aliquots of the collected fractions wereboiled in the presence of DTT and blotted in duplicate ontonitrocellulose filters. The filters were incubated with anti-HPV-6 andanti-HPV-16 specific Mabs which react with denatured L1 (8, 9),revealing that the two L1 proteins were enriched in the same fractions(FIG. 6A, a and c). The dot-blot experiment was repeated withoutdenaturing and without reducing the protein samples and using anti-HPV-6and HPV-16 L1 specific Mabs which were previously reported to reactexclusively with intact VLPs in enzyme-linked immunosorbent assay(ELISA) experiments (8, 9). The result obtained confirmed that the twoconformationally dependent Mabs were able to recognize the L1 proteinswhich copurified in the CsCl fractions (FIG. 6A, b and d). As expected,the two Mabs reacted specifically with HPV-6 and HPV-16 control VLPsonly under nondenaturing and nonreducing conditions (FIG. 6A, e).Western blot analysis of fraction 5 confirmed that both HPV-6 and HPV-16L2 proteins were also present (FIG. 6B, a and b). Estimation of therefractive index of the identified protein peak gave a value of 129-1.3mg/ml. EM analysis of the enriched fraction revealed the presence ofVLPs which appeared to be similar to control VLPs formed by either HPV-6or HPV-16 L1 (FIG. 7).

To evaluate whether the HPV-6 and HPV-16 L1 proteins could interact andassemble into mosaic VLPs, we performed immunoprecipitation experimentsusing CsCl banded VIPs and the specific anti-HPV-6 L1 conformationallydependent Mab H6.B10.5 (9). Approximately 1 μg of CsCL banded VLPs werediluted with PBS and incubated with the conformationally dependentanti-HPV-6 L1 Mab H6.B 10.5 (1:1000 dilution) overnight at 4° C. withgentle shaking. The immune complexes were collected with Protein ASepharose CL-4B (Pharmacia Biotech), washed 4 times with 1 ml PBS,suspended in sample buffer, boiled for 5 minutes, subjected to SDS-PAGEand analyzed by Western blot using anti-HPV6 and anti-HPV-16 L1 Mabs.The Western blot carried out on the immunoprecipitates usingtype-specific anti-L1 Mabs (FIG. 8) identified three major bands: (A)was a Mab-derived band, since it could be also observed when theconformational Mab was immunoblotted with the anti-mouse antibody; (B)was a band that appeared only when the VLPs were incubated with theconformational anti-HPV-6 L1 Mab (lanes 3), identifying specificallyimmunoprecipitated proteins with an electrophoretic mobilitycorresponding to that of HPV-6 L1 (a, lane 4) and HPV-16 L1 (b, lane 5);(C) was a resin-derived band that was also detected when an aliquot ofprotein A Sepharose was suspended in PBS and immunoblotted with theanti-mouse antibody. Bands (B) were not visible when theimmunoprecipitation was carried out using an unrelated Mab Similarly,HPV-16 L1 could not be detected when HPV-6 and HPV-16 VLPs were mixedand immunoprecipitated.

Example 6 Mouse Immunization with VLPs

To investigate whether HPV-6/16 mosaic VLPs were able to induce animmune response directed against both HPV types, groups of mice wereimmunized subcutaneously with HPV-6, HPV-16 and mosaic VLPs and the serawere tested after the third immunization. Six week old female Balb/cmice were injected subcutaneously with 20 μg of the following purifiedantigens: (I) HPV-6 VLPs, (ii) HPV-16 VLPs, (iii) HPV-6/16 VLPs. All theantigens were administered with equal volume of MF59 adjuvant (30). Agroup of control mice was injected only with MF59. The mice were boostedwith 15 μg of the respective antigen at week 3 and 10 μg at week 5.Serum samples were collected on day 12 after the final booster andassayed for capsid protein specific antibodies.

FIG. 9A shows the result of the Western blot carried out with the threetypes of denatured VLPs incubated with three sera, each representativeof the different groups of immunized mice. While the reactivity of thesera from mice immunized either with HPV-6 or HPV-16 VLPs waspredominantly type-specific (FIG. 9A, a and b), the serum from mouse 16(S16), immunized with HPV6/16 VLPs, reacted against both HPV-6 andHPV-16 L1 (FIG. 9A, c). To analyze whether the immune response was alsodirected against conformational epitopes of the L1 proteins, equalamounts of either HPV-6 or HPV-16 VLPs were blotted under denaturing andnondenaturing conditions and incubated with the 516 antiserum. FIG. 9Bshows that the signal was significantly lower when the samples weredenatured and reduced, suggesting that conformational antibodies hadbeen elicited.

The foregoing examples are meant to illustrate the invention and are notto be construed to limit the invention in any way. Those skilled in theart will recognize modifications that are within the spirit and scope ofthe invention. All references cited herein are hereby incorporated byreference in their entirety.

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1. A diploid cell comprising vectors for expressing capsid proteins fromat least two Human Papilloma Viruses (HPV), wherein said at least twoHPVs are HPV type 6 and HPV type 16, wherein the capsid proteinscomprise a late 1 (L1) capsid protein from HPV type 6, an L1 capsidprotein from HPV type 16, a minor capsid protein late 2 (L2) from HPVtype 6 and an L2 capsid protein from HPV type
 16. 2. The diploid cell ofclaim 1, wherein the cell is a yeast cell.
 3. The diploid cell of claim2, wherein the yeast is Saccharomyces cerevisiae.
 4. A method forproducing a diploid cell according to claim 1 comprising: a) cloningsaid capsid proteins into expression cassettes comprising the samepromoters and termination sequences; and b) expressing said cassettes inthe same diploid cell.
 5. The method of claim 4, wherein the diploidcell is a yeast cell.
 6. The method of claim 5, wherein the yeast isSaccharomyces cerevisiae.
 7. The method of claim 4, wherein the L1expression cassettes are cloned into non-integrative vectors, and the L2expression cassettes are cloned into integrative vectors.
 8. The methodof claim 7 wherein the non-integrative vector is pBS24.1.
 9. The methodof claim 7 wherein the integrative vector is pUC8.
 10. A method forproducing a virus-like particle (VLP) comprising: (a) cloning capsidproteins that comprise a late 1 (L1) capsid protein from HPV type 6, anL1 capsid protein from HPV type 16, a minor capsid protein late 2 (L2)from HPV type 6 and an L2 capsid protein from HPV type 16 intoexpression cassettes comprising the same promoters and terminationsequences; and (b) expressing the cassettes in the same diploid cell.11. The method of claim 10 wherein the diploid cell is a yeast cell. 12.The method of claim 11 wherein the yeast is Saccharomyces cerevisiae.13. The method of claim 10 wherein said L1 expression cassettes arecloned into non-integrative vectors, and said L2 expression cassettesare cloned into integrative vectors.
 14. The method of claim 13 whereinthe non-integrative vector is pBS24.1.
 15. The method of claim 13wherein the integrative vector is pUC8.